The heat stress response is associated with several beneficial adaptations that promote cell health and survival. Specifically, in vitro and animal investigations suggest that repeated exposures to a mild heat stress (~40°C) elicits positive mitochondrial adaptations in skeletal muscle comparable to those observed with exercise. To assess whether such adaptations translate to human skeletal muscle, we produced local, deep tissue heating of the vastus lateralis via pulsed shortwave diathermy in 20 men (n=10) and women (n=10). Diathermy increased muscle temperature by 3.9°C within 30 minutes of application. Immediately following a single 2-hr heating session, we observed increased phosphorylation of AMPK and ERK1/2, but not of p38 MAPK nor JNK. Following repeated heat exposures (2-hr daily for 6 consecutive days), we observed a significant cellular heat stress response, as heat shock protein 70 and 90 increased 45% and 38%, respectively. In addition, PGC-1α and mitochondrial electron transport protein complexes I and V expression were increased after heating. These increases were accompanied by augmentation of maximal coupled, and uncoupled, respiratory capacity, measured via high-resolution respirometry. Our data provide the first evidence that mitochondrial adaptation can be elicited in human skeletal muscle in response to repeated exposures to mild heat stress.